Growth factor complex

ABSTRACT

An isolated protein complex is provided which includes a growth factor, growth factor binding protein and vitronectin. Preferably, the isolated protein complex includes an insulin-like growth factor-I, insulin-like growth factor binding protein-3 or insulin-like growth factor binding protein-5 and vitronectin. Also provided are methods of modulating cell proliferation and/or migration by administering said protein complex for the purposes of wound healing, skin repair and tissue replacement therapy. Conversely, by using agents that disrupt growth factor protein complexes formed in vivo, growth factor-driven cell proliferation and/or migration may be suppressed such as for the purposes of treating cancers, psoriasis, atherosclerosis and wounds prone to hypertrophic scarring.

FIELD OF THE INVENTION

[0001] THIS INVENTION relates to an isolated protein complex whichincludes a growth factor binding protein and vitronectin. In particular,this invention relates to an isolated protein complex which includes aninsulin-like growth factor, an insulin-like growth factor bindingprotein and vitronectin. Also provided by the invention are growthfactor complexes comprising variant growth factors and/or growth factorbinding proteins that facilitate or enhance formation of the growthfactor complexes. This invention also provides methods of modulatingcell proliferation and/or migration by administering said proteincomplex for the purposes of wound healing, skin repair, cosmetic skinmaintenance and tissue replacement therapy. Conversely, by disruptingprotein complexes formed in vivo, growth factor-driven cellproliferation and/or migration can be suppressed such as for thepurposes of treating cancers, psoriasis, atherosclerosis and woundsprone to hypertrophic scarring. These treatments may have medical andveterinary applications.

BACKGROUND OF THE INVENTION

[0002] Skin growth, repair and healing are subject to complex biologicalcontrol mechanisms which act via both positive and negative signals.Such signals act at the level of controlling cell proliferation,differentiation and migration, and are typically mediated by growthfactor polypeptides. In this regard, important growth factors includeepidermal growth factor (EGF) and insulin-like growth factors (IGF-I and-II).

[0003] Human IGF-I has been reported to exert a wide range of biologicalactivities including stimulation of cell proliferation, differentiationand migration, protection from protein degradation and apoptosis, aswell as regulation of endocrine factors such as growth hormone. IGF-IFhas similar properties to IGF-I but appears to be more relevant tocarcinogenesis and fetal and embryonic development, IGF-I having agreater role in postnatal development.

[0004] Both IGF-I and IGF-II act through a binding interaction with thetype I IGF receptor (IGFR). The availability of the IGFs for such aninteraction is regulated by insulin like growth factor binding proteins(IGFBPs 1-6). IGFBPs are known to both positively and negativelyregulate IGF function as well as exhibit IGF-independent activity.

[0005] Another functional component of IGF pathways is the type II IGFRwhich is also known as the cation-independent mannose-6-phosphatereceptor (CI-MPR). The type II IGFR is a multifunctional protein thatbinds lysosomal enzymes bearing mannose-6-phosphate moieties, as well asIGF-II, although the functional significance of IGF-II binding isunclear (O'Dell & Day, 1998, Int. J. Biochem. Cell Biol. 30 767;Braulke, 1999, Horm. Metab. Res. 31 242; Nykjaer et al., 1998, J. Cell.Biol. 141 815).

[0006] The IGFs have also been reported to bind another group ofproteins termed “IGFBP-related proteins” which share structuralsimilarity and include connective tissue growth factor (CTGF) andproducts encoded by the mac25, nov and cyr61 genes. These bind IGFs withmuch lower affinity than do IGFBPs.

[0007] More recently, vitronectin (VN) has been identified as anextracellular matrix protein, structurally unrelated to IGFBPs andIGFBP-related proteins, that binds IGF-II but not IGF-I (Upton et al.,1999, Endocrinol. 140 2928).

[0008] Vitronectin is an ˜75 kD, glycosylated extracellular matrixprotein which is also found in blood, and has been implicated incancers, bone diseases and pathological disorders involving angiogenesis(reviewed in Schvartz et al., 1999, Int. J. Biochem. Cell Biol. 31 539).The role of vitronectin in events such as angiogenesis and tumorigenesisat least partly resides in the ability of vitronectin to bind integrinsand to interact with components of the urokinase plasminogen activatorsystem (for example PAI-1, uPAR, plasminogen) to thereby promote cellproliferation, adhesion, spreading and migration. Vitronectin has morespecifically been implicated in preventing tumor cell apoptosis inresponse to drug treatment (Uhm et al., 1999, Clin. Cancer Res. 5 1587).Vitronectin appears to be a carrier of IGF-II in the circulation(McMurtry et al., 1996, J. Endocrinol. 150 149).

OBJECT OF THE INVENTION

[0009] The present inventors have surprisingly discovered that IGFBPsbind vitronectin, and that IGF-1 can bind vitronectin when bound to anIGFBP.

[0010] It is therefore an object of the invention to provide an isolatedIGFBP and vitronectin-containing complex.

SUMMARY OF THE INVENTION

[0011] In a first aspect, the invention provides an isolated polypeptidecomplex comprising a growth factor binding protein and vitronectin.

[0012] Preferably, the isolated polypeptide complex farther comprises agrowth factor.

[0013] A preferred growth factor is insulin-like growth factor-I(IGF-I).

[0014] Other growth factors include epidermal growth factor (EGF),fibroblast growth factor. (FGF), basic fibroblast growth factor (bFGF),osteopontin, PAI-1 and transferrin.

[0015] Other biologically active proteins that may form proteincomplexes of the invention include thrombospondin-1, tenascin-C, PAI-1,plasminogen, fibrinogen, and fibrin.

[0016] A preferred growth factor binding protein is an insulin-likegrowth factor binding protein selected from the group consisting ofIGFBP1, IGFBP2, IGFBP3, IGFBP4, IGFBP5 and IGFBP6.

[0017] A more preferred insulin-like growth factor binding protein isIGFBP2, IGFBP3, IGFBP4 or IGFBP5.

[0018] An even more preferred insulin-like growth factor binding proteinis IGFBP3 or IGFBP5.

[0019] In a second aspect, the invention provides an isolated proteincomplex comprising an IGFBP-related protein and vitronectin.

[0020] Preferably, the isolated polypeptide complex further comprises agrowth factor, preferably IGF-I.

[0021] In one embodiment, the IGFBP-related protein is selected from thegroup consisting of connective tissue growth factor (CTGF), apolypeptide encoded by the mac25 gene, a polypeptide encoded by the novgene and a polypeptide encoded by the cyr61 gene.

[0022] In a third aspect, the invention provides a isolated proteincomplex comprising vitronectin, a variant growth factor and/or a variantgrowth factor binding protein.

[0023] In one embodiment, the isolated protein complex of this aspectcomprises vitronectin and a variant growth factor engineered to includea heparin binding domain (HBD).

[0024] In another embodiment, the isolated protein complex of thisaspect comprises vitronectin and a non-glycosylated growth factorbinding protein.

[0025] In yet another embodiment, the isolated protein complex of thisaspect comprises vitronectin and a variant growth factor selected fromthe group consisting of des(1-6)IGF-II and des(1-3)IGF-I.

[0026] Also contemplated according to the first-, second- andthird-mentioned aspects is that the isolated protein complex of theinvention may further include an acid-labile subunit, a polypeptidewhich can complex with an IGFBP, referred to hereinafter as ALS.

[0027] The invention according to the first-, second- andthird-mentioned aspects also contemplates isolated protein complexescomprising variants and biologically-active fragments of growth factors,growth factor binding proteins, IGFBP-related proteins and vitronectin,and use of such complexes. Biologically-active fragments and variantsinclude within their scope analogues, mutants, agonists and antagonistsof said growth factors, growth factor binding proteins, IGFBP-relatedproteins and vitronectin.

[0028] In a fourth aspect, the invention provides a pharmaceuticalcomposition comprising one or more isolated protein complexes accordingto the first-, second- or third-mentioned aspect and apharmaceutically-acceptable carrier or diluent.

[0029] In a fifth aspect, the invention provides a pharmaceuticalcomposition comprising a expression construct comprising one or morenucleic acids encoding an isolated protein complex according to thefirst-, second- or third-mentioned aspect and apharmaceutically-acceptable carrier or diluent

[0030] In a sixth aspect, the invention provides a transformed cellcapable of expressing a recombinant protein complex, or recombinantproteins capable of forming said complex, according to the first-,second- or third-mentioned aspects.

[0031] In a seventh aspect, the invention provides a method ofmodulating cell proliferation and/or migration including the step ofadministering to an animal or isolated cells thereof, an isolatedprotein complex according to the first-, second- or third-mentionedaspects.

[0032] Preferably, the isolated protein complex comprises an IGFBP andvitronectin.

[0033] Preferably, the isolated protein complex further comprises IGF-I.

[0034] In an eighth aspect, the invention provides a method ofmodulating cell proliferation and/or migration, including the step ofadministering to an animal or isolated cells thereof an agent whichprevents or disrupts formation of a protein complex according to thefirst-, second- or third-mentioned aspects.

[0035] Preferably, the agent prevents or disrupts an interaction betweenan IGFBP and vitronectin.

[0036] More preferably, the agent prevents or disrupts an interactionbetween IGFBP and vitronectin wherein the protein complex comprisesIGF-I.

[0037] The agent may be an antagonist of an interaction between an IGFBPand vitronectin or between an IGFBP-related protein and vitronectin, forexample.

[0038] An example of an agent that inhibits formation of IGFBP andvitronectin complexes is IGF-II.

[0039] As will be described in more detail hereinafter, disruption ofinteractions between vitronectin and IGFBPs may not only inhibit tumourcell proliferation, but also inhibit tumour metastasis, both eventsbeing central to tumour pathology.

[0040] Further aspects of the invention provide uses of the isolatedprotein complexes and methods according to the aforementioned aspects ofthe invention in therapeutic or prophylactic treatments of diseasesinvolving epithelial cells such as psoriasis, atherosclerosis,deterioration of the gastrointestinal epithelium and epithelial breastcancer, and/or in treatments which promote wound healing, skin repair,ulcer and burn healing, in vitro skin regeneration such as for graftingof autologous skin, bone regeneration and repair of damaged neuronaltissue.

[0041] Accordingly, the invention also provides a surgical implant orprosthesis comprising an isolated protein complex of the invention. Thesurgical implant or prosthesis may be coated, impregnated or otherwisepretreated with said isolated protein complex.

[0042] The animal treated according to the invention may be a mammal,preferably a human, or may be a non-mammalian vertebrate such as a fish,reptile or bird, or cells isolated from any of these.

[0043] Throughout this specification, unless otherwise indicated,“comprise”, “comprises” and “comprising” are used inclusively ratherthan exclusively, so that a stated integer or group of integers mayinclude one or more other non-stated integers or groups of integers.

BRIEF DESCRIPTION OF THE FIGURES AND TABLES

[0044] TABLE 1: List of references disclosing nucleic acids encodinggrowth factors and growth factor binding proteins.

[0045] TABLE 2: IGF-I and IGFBP-5 bound to vitronectin stimulatesprotein synthesis in HaCAT human keratinocytes. Data are derived frommeasurements of ³H-leucine incorporation and are expressed as %stimulation above control (without IGF-I, IGFBP-5 or vitronectin) over24 hr from a single experiment in which each treatment was tested intriplicate. IGF-I was added to IGFBP-5 (5 ng/well) in the presence (+)of vitronectin (300 ng/well) or in the absence (−) of vitronectin.

[0046]FIG. 1: Competition binding assay using increasing concentrationsof either insulin (▴) or IGF-II (♦) to compete with [¹²⁵I]-IGF-II forbinding to 300 ng vitronectin (VN) per well. Radiolabeled IGF-II (10,000cpm) was added to VN-coated wells and the number of counts bound wasdetermined after overnight incubation and several washes. The binding isexpressed as a percentage of the binding observed in control wells withno IGF-II or insulin added. The results are shown as the average ofthree replicates ± standard deviation from a representative of threeexperiments.

[0047]FIG. 2: Competition binding assay using increasing concentrationsof either IGB-I or IGF-II to compete with [(¹²⁵I]-IGF-II for binding to300 ng vitronectin (VN) per well. Radiolabeled IGB-II (10,000 cpm) wasadded to either VN (▪)- or IGFBP2 (♦)-coated wells and the number ofcounts bound was determined after overnight incubation and severalwashes. The binding is expressed as a percentage of the binding observedin control wells with no IGF added.

[0048]FIG. 3: Competition binding assay using increasing concentrationsof either proIGF-II (▪) or IGF-II (♦) to compete with [¹²⁵I]-IGF-II forbinding to 300 ng vitronectin VN) per well. Radiolabeled IGF-II (10,000cpm) was added to vitronectin-coated wells and the number of countsbound determined after an overnight incubation and several washes. Thebinding is expressed as a percentage of the binding observed in thecontrol wells with no added IGF-II or proIGF-II. The results are shownas the average of three replicates 4 SEM from two separate experiments.

[0049]FIG. 4: Competition binding assay using increasing concentrationsof either PAI-1 (▪) or IGF-II (♦) to compete with [¹²⁵I]-IGF-II forbinding to 300 ng of vitronectin (VN) per well. Radiolabeled IGF-II(10,000 cpm) was added to vitronectin-coated well and the number ofcounts bound was determined after an overnight incubation and severalwashes. The binding is expressed as a percentage of the binding observedin the control wells with no added IGF-II or PAI-1. The results areshown as the average of three replicates ± standard deviation from arepresentative of three separate experiments.

[0050]FIG. 5: Competition binding assay comparing effect ofpreincubation of (A) IGF-II and IGFBP3 and (1) IGF-I and IGFBP3 uponbinding to vitronectin. IGFBP3 was non-glycosylated and produced in E.coli. Increasing concentrations of IGFBP3 plus either 10,000 cpm[¹²⁵I]-IGF-II (A) or [¹²⁵I]-IGF-I (B) were preincubated for 4 hr andadded to vitronectin-coated wells. Alternatively, IGFBP3 plus either10,000 cpm [¹²⁵I]-IGF-II (A) or [¹²⁵I]-IGF-I (B) were added tovitronectin-coated wells without preincubation The binding is expressedas the cpm obtained in the absence of non-specific binding. The resultsare shown as the average of three replicates from a representative ofthree separate experiments.

[0051]FIG. 6: Binding of labelled IGF-I to VN-coated wells in thepresence of: (A) IGFBP1, (B) IGFBP2, (C) IGFBP3, (D) IGFBP4, (E) IGFBP5and (F) IGFBP6. The recombinant IGFBPs were produced in mammalian cells.The data, expressed as average cpm of labelled IGF-I bound/VN-coatedwell (300 ng/well) in the presence of the indicated IGFBP arc from sixindividual determinations. Ten thousand cpm of radiolabelled IGF-I wasadded to each well.

[0052]FIG. 7: Binding of labelled IGF-I to VN in then presence of “GlyIGFBP-3” (glycoslyated IGFBP-3), “IGFBP-3 HBD mutant” (IGFBP-3 with theputative heparin binding domain mutated) and to “non-gly IGFBP-3”(non-glycosylated IGFBP-3).

[0053]FIG. 8: Competition binding assay using increasing concentrationsof IGFs and desIGFs to compete with non-glycosylated IGFBP3 incubatedwith [¹²⁵I]-IGF-I or -II 30 ng (A) or 10 ng (B) of IGFBP3 plus 10,000cpm [125I]-IGF-II (A) or IGF-I (B) were added to VN-coated wells.Increasing concentrations of IGF-I, IGF-I, des (1-3) IGF-I (not shown)and des(1-6) IGF-II were added to compete with the radiolabel forbinding to VN. Control wells coated with VN were treated with either 10ng (A) or 30 ng (B) IGFBP3 are shown together with control wells coatedwith VN alone. The binding is expressed as cpm obtained in the absenceof non-specific binding. The results are expressed as the average ofthree replicates ± standard deviation from a representative of threeseparate experiments.

[0054]FIG. 9: Stimulation of protein synthesis in human keratinocytes.The data, expressed as % stimulation above control (-VN, -IGF-II) over24 h is pooled from three replicate experiments in which each treatmentwas tested in triplicate. The theoretical additive effect represented byopen bars (effect of VN alone) combined with grey bars (effect of IGF-IIalone) is compared with the actual observed effect of IGF-II prebound toVN (black bars). In all instances except the lowest concentration ofIGF-II tested, the actual observed effect is significantly greater(p<0.05) than the calculated additive effect.

[0055]FIG. 10: Binding of labelled IGF-II to VN-coated wells in thepresence of: (A) IGFBP1, (B) IGFBP2, (C) IGFBP3, (D) IGFBP4, (E) IGFBP5and (F) IGFBP6. The recombinant IGFBPs were produced in mammalian cells.The data, expressed as average cpm of labelled IGF-II bound/VN-coatedwell (300 ng/well) in the presence of the indicated IGFBP are from sixindividual determinations. Ten thousand cpm of radiolabelled IGF-II wasadded to each well.

DETAILED DESCRIPTION OF THE INVENTION

[0056] The present invention has arisen, at least in part, from thediscovery by the present inventors that IGF-I binds vitronectin via abinding interaction between an IGFBP and vitronectin. Furthermore, thepresent invention describes variant IGFs and IGFBPs that may be used toaugment or diminish binding between IGFS, IGFBP, and vitronectin. Thesediscoveries have led the present inventors to manipulate these bindinginteractions in vitro with a view to manipulating contingent in vivobiological events associated with cell growth, proliferation andmigration. This invention therefore has utility in medical treatmentssuch as wound healing, skin repair and maintenance, bone regeneration,atherosclerosis and cancer therapy in both medical and veterinary areas.

[0057] For the purposes of this invention, by “isolated” is meantremoved from a natural state or otherwise subjected to humanmanipulation. Isolated material may be substantially or essentially freefrom components that normally accompany it in its natural state, or maybe manipulated so as to be in an artificial state together withcomponents that normally accompany it in its natural state.

[0058] By “polypeptide” is also meant “protein”, either term referringto an amino acid polymer.

[0059] Proteins and peptides inclusive of growth factor, growth factorbinding proteins and vitronectin proteins may be isolated in native,chemical synthetic or recombinant synthetic form.

[0060] A “peptide” is a protein having no more than fifty (50) aminoacids.

[0061] A “biologically-active fragment” is a fragment, portion orsegment of a protein which displays at least 1%, preferably at least10%, more preferably at least 25% and even more preferably at least 50%of the biological activity of the protein.

[0062] Peptides may be readily synthesized by recombinant or chemicalsynthesis techniques. For example, reference may be made to solutionsynthesis or solid phase synthesis as described, for example, in Chapter9 entitled “Peptide Synthesis” by Atherton and Shephard which isincluded in a publication entitled “Synthetic Vaccines” edited byNicholson and published by Blackwell Scientific Publications. Peptidesynthesis methods are also described in Chapter 18 of CURRENT PROTOCOLSIN PROTEIN SCIENCE Eds. Coligan et al. (John Wiley & Sons NY, 1997)which is incorporated herein by reference.

[0063] Alternatively, peptides can be produced by digestion of apolypeptide of the invention with proteinases such as endoLys-C,endoArg-C, endoGlu-C and staphylococcus V8-protease. The digestedfragments can be purified by, for example, high performance liquidchromatographic (HPLC) techniques.

[0064] In a preferred form, the invention provides an isolated proteincomplex comprising vitronectin, a growth factor and a growth factorbinding protein.

[0065] By “growth factor” is meant a molecule that stimulates orpromotes growth of an organism or cells of said organism. A preferredgrowth factor is a protein or peptide that stimulates cell division, andin particular, mammalian cell division.

[0066] Preferably, isolated protein complexes of the invention comprisethe growth factor IGF-I.

[0067] Isolated IGP and IGFBP polypeptides are commercially availablefrom sources such as GroPep (Adelaide, Australia), while VN polypeptidesare commercially available from sources such as Promega Corporation(Madison Wis. USA). Recombinant IGFs, IGFBPs and VN are readily made bypersons skilled in the art, as will be discussed in more detailhereinafter.

[0068] As will be appreciated by the skilled person, the invention alsoincludes precursor forms of IGFs. Examples of pro-IGF-I proteins areIGF-I proteins that have had the signal peptide removed but are notfully processed by cleavage of the E-domain. IGF-I has three different Edomains that result from differential mRNA splicing and these precursorproteins may be present in isolated protein complexes of the invention.

[0069] However, the invention also contemplates isolated proteincomplexes comprising a growth factor or other biologically activeprotein such as epidermal growth factor (EGF; (Heldin et al, 1981,Science 4 1122-1123), fibroblast growth factor (FGF; Nurcombe et al.,2000, J. Biol. Chem. 275 30009-30018), basic fibroblast growth factor(bFGF; Taraboletti et al., 1997, Cell Growth. Differ. 8 471479),osteopontin (Nam et al., 2000, Endocrinol. 141 1100), thrombospondin-1(Nam et al., 2000, supra), tenascin-C (Arai et al., 1996, J. Biol Chem.271 6099), PAI-1 (Nam et al., 1997, Endocrinol. 138 2972), plasminogen(Campbell et al., 1998, Am. J. Physiol. 275 E321), fibrinogen (Campbellet al., 1999, J. Biol. Chem 274 30215), fibrin (Campbell et al., 1999,supra) or transferrin (Weinzimer et al., 2001, J. Clin. Endocrinol.Metab. 86 1806).

[0070] Preferably, isolated protein complexes comprising osteopontinthrombospondin-1, tenascin-C or PAI-1 further comprise IGFBP-5.

[0071] Preferably, isolated protein complexes comprising plasminogenfibrinogen, fibrin or transferrin further comprise IGFBP-3.

[0072] The invention contemplates isolated protein complexes comprisingmonomeric and multimeric vitronectin, as vitronectin can exist inmonomeric and multimeric states. In particular, multimeric VNaccumulates in areas of vascular injury and also is the predominant formof VN in tissue. Thus the multimeric form of VN provides the opportunityto form a VN complex in which more than one type of growth factor orgrowth factor binding protein can be delivered at the same time.

[0073] It will also be appreciated by the skilled person that isolatedprotein complexs of the invention may include vitronectin in “native”,“denatured” or “extended” states as are well undestood in the art.

[0074] The invention also contemplates isolated growth factor complexescomprising nectinepsin, which is an extracellular matrix protein thatshows 60% homology to VN at the amino acid level (Blanchert et al.,1996, J. Biol. Chem. 271 26220-26226).

[0075] It will also be understood that variants of growth factors,growth factor binding proteins and/or vitronectin may be used to formisolated protein complexes of the invention and may be useful in themethods of use set forth herein.

[0076] As used herein, “variant” proteins, polypeptides and peptides ofthe invention include those in which one or more amino acids have beenreplaced by different amino acids.

[0077] It is well understood in the art that some amino acids may bechanged to others with broadly similar properties without changing thenature of the activity of the polypeptide (conservative substitutions).

[0078] Substantial changes in function may be made by selectingsubstitutions that are less conservative. Other replacements would benon-conservative substitutions and relatively fewer of these may betolerated. Generally, the substitutions which are likely to produce thegreatest changes in a polypeptide's properties are those in which (a) ahydrophilic residue (e.g., Ser or Thr) is substituted for, or by, ahydrophobic residue (e.g., Ala, Leu, Ile, Phe or Val); (b) a cysteine orproline is substituted for, or by, any other residue; (c) a residuehaving an electropositive side chain (e.g., Arg, His or Lys) issubstituted for, or by, an electronegative residue (e.g., Glu or Asp) or(d) a residue having a bulky side chain (e.g., Phe or Trp) issubstituted for, or by, one having a smaller side chain (e.g., Ala, Ser)or no side chain (e.g., Gly).

[0079] Variants also include proteins, polypeptides and peptides whichhave been altered, for example by conjugation or complexing with otherchemical moieties or by post-translational modification techniques aswould be understood in the art. Such derivatives include amino aciddeletions and/or additions.

[0080] Other polypeptide and peptide variants contemplated by theinvention include, but are not limited to, modification to side chains,incorporation of unnatural amino acids and/or their derivatives duringpeptide, polypeptide or protein synthesis and the use of crosslinkersand other chemicals which impose conformational constraints on thepolypeptides and peptide variants of the invention. Examples of sidechain modifications contemplated by the present invention includemodifications of amino groups such as by acylation with aceticanhydride; acylation of amino groups with succinic anhydride andtetrahydrophthalic anhydride; amidination with methylacetimidate;carbamoylation of amino groups with cyanate; pyridoxylation of lysinewith pyridoxal-5-phosphate followed by reduction with NaBH₄; reductivealkylation by reaction with an aldehyde followed by reduction withNaBH₄; and trinitrobenzylation of amino groups with2,4,6-trinitrobenzene sulphonic acid (TNBS).

[0081] The carboxyl group may be modified by carbodiimide activation viaO-acylisourea formation followed by subsequent derivitization, by way ofexample, to a corresponding amide.

[0082] The guanidine group of arginine residues may be modified byformation of heterocyclic condensation products with reagents such as2,3-butanedione, phenylglyoxal and glyoxal.

[0083] Sulphydryl groups may be modified by methods such as performicacid oxidation to cysteic acid; formation of mercurial derivatives using4-chloromercuriphenylsulphonic acid, 4-chloromercuribenzoate;2-chloromercuri-4-nitrophenol, phenylmercury chloride, and othermercurials; formation of a mixed disulphides with other thiol compounds;reaction with maleimide, maleic anhydride or other substitutedmaleimide; carboxymethylation with iodoacetic acid or iodoacetamide; andcarbamoylation with cyanate at alkaline pH.

[0084] Tryptophan residues may be modified, for example, by alkylationof the indole ring with 2-hydroxy-5-nitrobenzyl bromide or sulphonylhalides or by oxidation with N-bromosuccinimide.

[0085] Tyrosine residues may be modified by nitration withtetranitromethane to form a 3-nitrotyrosine derivative.

[0086] The imidazole ring of a histidine residue may be modified byN-carbethoxylation with diethylpyrocarbonate or by alkylation withiodoacetic acid derivatives.

[0087] Examples of incorporating unnatural amino acids and derivativesduring peptide synthesis include but are not limited to, use of 4-aminobutyric acid, 6-aminohexanoic acid, 4-amino-3-hydroxy-5-phenylpentanoicacid, 4-amino-3-hydroxy-6-methylheptanoic acid, t-butylglycine,norleucine, norvaline, phenylglycine, ornithine, sarcosine, 2-thienylalanine and/or D-isomers of amino acids.

[0088] Modifications also include within their scope O- and N-linkedglycosylation variants and non-glycosylated forms of proteins that, intheir naturally-occurring state, are glycosylated.

[0089] With regard to variants, these may be created by mutagenizing apolypeptide or by mutagenizing an encoding nucleic acid, such as byrandom mutagenesis or site-directed mutagenesis. Examples of nucleicacid mutagenesis methods are provided in in Chapter 9 of CURRENTPROTOCOLS IN MOLECULAR BIOLOGY, Ausubel et al., supra which isincorporated herein by reference.

[0090] It will be appreciated by the skilled person that site-directedmutagenesis is best performed where knowledge of the amino acid residuesthat contribute to biological activity is available. In many cases, thisinformation is not available, or can only be inferred by molecularmodelling approximations, for example.

[0091] In such cases, random mutagenesis is contemplated. Randommutagenesis methods include chemical modification of proteins byhydroxylamine (Ruan et al., 1997, Gene 188 35), incorporation of dNTPanalogs into nucleic acids (Zaccolo et al., 1996, J. Mol. Biol. 255 589)and PCR-based random mutagenesis such as described in Stemmer, 1994,Proc. Natl. Acad. Sci. USA 91 10747 or Shafikhani et al., 1997,Biotechniques 23 304, each of which references is incorporated herein.It is also noted that PCR-based random mutagenesis kits are commerciallyavailable, such as the Diversify™ kit (Clontech).

[0092] The invention also contemplates use of growth factor variantssuch as des(1-6)IGF-II and des(1-3)IGF-I to form isolated proteincomplexes of the invention.

[0093] Other variant IGFs and IGFBPs useful as agonists or antagonistswill be described in more detail hereinafter.

[0094] Recombinant Growth Factor Complexes

[0095] It will be appreciated that isolated protein complexes may beproduced using recombinant growth factors, growth factor bindingproteins and/or vitronectin, by expression of an encoding nucleic acidin an appropriate host cell or in a cell-free expression system as arewell known in the art.

[0096] The term “nucleic acid” as used herein designates single- ordouble-stranded mRNA, RNA, cRNA and DNA, said DNA inclusive of cDNA andgenomic DNA.

[0097] A “polynucleotide” is a nucleic acid having eighty (80) or morecontiguous nucleotides, while an “oligonucleotide” has less than eighty(80) contiguous nucleotides.

[0098] A “probe” may be a single or double-stranded oligonucleotide orpolynucleotide, suitably labeled for the purpose of detectingcomplementary sequences in Northern or Southern blotting, for example.

[0099] A “primer” is usually a single-stranded oligonucleotide,preferably having 15-50 contiguous nucleotides, which is capable ofannealing to a complementary nucleic acid “template” and being extendedin a template-dependent fashion by the action of a DNA polymerase suchas Taq polymerase, RNA-dependent DNA polymerase or Sequenase™.

[0100] Nucleic acids encoding IGFs, EGF, IGFBPs, ALS and VN are well 10known in the art and have been available for many years. However, theskilled person is referred to Table 1 which lists references thatprovide examples of these nucleic acid sequences. All references inTable I are incorporated herein by reference.

[0101] Nucleic acids useful according to the present invention may beprepared according to the following procedure:

[0102] (i) creating primers which are, optionally, degenerate whereineach comprises a respective portion of a target nucleic acid; and

[0103] (ii) using said primers in combination with a nucleic acidamplification technique to amplify one or more amplification productsfrom a nucleic acid extract.

[0104] Suitable nucleic acid amplification techniques are well known tothe skilled person, and include polymerase chain reaction (PCR) as forexample described in Chapter 15 of Ausubel et al. supra, which isincorporated herein by reference; strand displacement amplification(SDA) as for example described in U.S. Pat. No 5,422,252 which isincorporated herein by reference; rolling circle replication (RCR) asfor example described in Liu et al., 1996, J. Am. Chem. Soc. 118 1587,International application WO 92/01813 and International Application WO97/19193 which are incorporated herein by reference; nucleic acidsequence-based amplification (NASBA) as for example described bySooknanan et al.,1994, Biotechniques 17 1077 which is incorporatedherein by reference; ligase chain reaction (LCR) as for exampledescribed in International Application WO89/09385 which is incorporatedby reference herein; and Q-β replicase amplification as for exampledescribed by Tyagi et al., 1996, Proc. Natl. Acad. Sci. USA 93 5395which is incorporated herein by reference.

[0105] As used herein, an “amplification product” refers to a nucleicacid product generated by nucleic acid amplification techniques.

[0106] Recombinant proteins may be prepared by any suitable procedureknown to those of skill in the art.

[0107] For example, the recombinant protein may be prepared by aprocedure including the steps of:

[0108] (i) preparing an expression construct which comprises a nucleicacid, operably linked to one or more regulatory nucleotide sequences;

[0109] (ii) transfecting or transforming a suitable host cell with theexpression construct; and

[0110] (iii) expressing the polypeptide in said host cell.

[0111] For the purposes of host cell expression, the recombinant nucleicacid is operably linked to one or more regulatory sequences in anexpression vector.

[0112] An “expression vector” may be either a self-replicatingextra-chromosomal vector such as a plasmid, or a vector that integratesinto a host genome.

[0113] By “operably linked” is meant that said regulatory nucleotidesequence(s) is/are positioned relative to the recombinant nucleic acidof the invention to initiate, regulate or otherwise controltranscription.

[0114] Regulatory nucleotide sequences will generally be appropriate forthe host cell used for expression. Numerous types of appropriateexpression vectors and suitable regulatory sequences are known in theart for a variety of host cells.

[0115] Typically, said one or more regulatory nucleotide sequences mayinclude, but are not limited to, promoter sequences, leader or signalsequences, ribosomal binding sites, transcriptional start andtermination sequences, translational start and termination sequences,and enhancer or activator sequences.

[0116] Constitutive or inducible promoters as known in the art arecontemplated by the invention. The promoters may be either naturallyoccurring promoters, or hybrid promoters that combine elements of morethan one promoter.

[0117] In a preferred embodiment, the expression vector contains aselectable marker gene to allow the selection of transformed host cells.Selectable marker genes are well known in the art and will vary with thehost cell used.

[0118] The expression vector may also include a fusion partner(typically provided by the expression vector) so that the recombinantpolypeptide of the invention is expressed as a fusion polypeptide withsaid fusion partner. The main advantage of fusion partners are that theyassist identification and/or purification of said fusion polypeptide andalso enhance protein expression levels and overall yeild.

[0119] In order to express said fusion polypeptide, it is necessary toligate a nucleotide sequence according to the invention into theexpression vector so that the translational reading frames of the fusionpartner and the nucleotide sequence of the invention coincide.

[0120] Well known examples of fusion partners include, but are notlimited to, glutathione-S-transferase (GST), Fc potion of human IgG,maltose binding protein (MBP) and hexahistidine (HIS₆), which areparticularly useful for isolation of the fusion polypeptide by affinitychromatography. For the purposes of fusion polypeptide purification byaffinity chromatography, relevant matrices for affinity chromatographyare glutathione-, amylose-, and nickel- or cobalt-conjugated resinsrespectively. Many such matrices are available in “kit” form, such asthe QIAexpress™ system (Qiagen) useful with (HIS₆) fusion partners andthe Pharmacia GST purification system.

[0121] Another fusion partner well known in the art is green fluorescentprotein (GFP). This fusion partner serves as a fluorescent “tag” whichallows the fusion polypeptide of the invention to be identified byfluorescence microscopy or by flow cytometry. The GFP tag is useful whenassessing subcellular localization of the fusion polypeptide of theinvention, or for isolating cells which express the fusion polypeptideof the invention. Flow cytometric methods such as fluorescence activatedcell sorting (FACS) are particularly useful in this latter application.

[0122] In some cases, the fusion partners also have a protease cleavagesite, such as for Factor X_(a) or Thrombin, which allow the relevantprotease to partially digest the fusion polypeptide of the invention andthereby liberate the recombinant polypeptide of the invention therefrom.The liberated polypeptide can then be isolated from the fusion partnerby subsequent chromatographic separation.

[0123] Fusion partners according to the invention also include withintheir scope “epitope tags”, which are usually short peptide sequencesfor which a specific antibody is available. Well known examples ofepitope tags for which specific monoclonal antibodies are readilyavailable include c-myc, influenza virus haemagglutinin and FLAG tags.

[0124] The recombinant protein may be conveniently prepared by a personskilled in the art using standard protocols as for example described inSambrook, et al., MOLECULAR CLONING. A Laboratory Manual (Cold SpringHarbor Press, 1989), incorporated herein by reference, in particularChapter 16 and 17; CURRENT PROTOCOLS IN MOLECULAR BIOLOGY Eds. Ausubelet al., (John Wiley & Sons, Inc. 1995-1999), incorporated herein byreference, in particular Chapters 10 and 16; and CURRENT PROTOCOLS INPROTEIN SCIENCE Eds. Coligan et al., (John Wiley & Sons, Inc. 1995-1999)which is incorporated by reference herein, in particular Chapters 1, 5and 6.

[0125] In one embodiment, recombinant expression of growth factor,growth factor binding protein and vitronectin may be performedseparately, and complexes formed therefrom.

[0126] In another embodiment, recombinant expression of growth factor,growth factor binding protein and vitronectin may be performed in thesame cell, and complexes formed therefrom.

[0127] As hereinbefore, polypeptides of the invention may be produced byculturing a host cell transformed with said expression constructcomprising a nucleic acid encoding a polypeptide, or polypeptidehomolog, of the invention. The conditions appropriate for proteinexpression will vary with the choice of expression vector and the hostcell. This is easily ascertained by one skilled in the art throughroutine experimentation.

[0128] Suitable host cells for recombinant expression include bacteriasuch as E. coli., Clostridium sp., Pseudomonas sp., yeast, plant cells,insect cells (such as Sf9) and mammalian cells such as fibroblasts andkeratinocytes.

[0129] Preferred host cells are human keratinocytes.

[0130] Inducible and non-inducible expression vectors are contemplated.In stably transfected mammalian cells, a number of inducible andrepressible systems have been devised including metallothionine (MT)inducible and tetracycline and repressible (tetR), each of which iscontemplated by the present invention.

[0131] Particular examples of suitable expression vectors and methods ofrecombinant IGFBP expression may be found in U.S. Pat. No. 5,973,115which is incorporated herein by reference.

[0132] Mimetics, Agonists and Antagonists

[0133] The invention contemplates agents which may promote, prevent ordisrupt formation of protein complexes comprising growth factors, growthfactor binding proteins and vitronectin. Such an agent may be a mimetic.The term “mimetic” is used herein to refer to molecules that aredesigned to resemble particular functional regions of proteins orpeptides, and includes within its scope the terms “agonist”, “analogue”and “antagonist” as are well understood in the art.

[0134] Of relevance is the elucidation of the portions of IGFs andIGFBPs which are responsible for IGF-IGFBP binding, as described inInternational Publication WO00/23469. Furthermore, agonist variants ofIGF-I have been made which selectively bind IGFBP-1 or IGFBP-3, asdescribed in International Publication WO00/40612.

[0135] It is therefore contemplated that agents could be engineeredwhich disrupt or prevent formation of polypeptide complexes betweenIGFBPs and VN. An example would be a peptide which competes for bindingof the IGFBP to VN by resembling the binding site on VN or the IGFBP.

[0136] As will be described in more detail hereinafter, IGF-II is anagent that can inhibit binding between an IGFBP and vitronectin. It isalso proposed that residues V³⁵ or S³⁶ along with S³⁹ of the RVSRRSRsequence at positions 34-40 in the C-domain of IGF-II could be deletedto thereby resemble the IIBD of IGFBP3 (BXBBB wherein B is a basic aminoacid residue). This would create an agent even more capable ofinhibiting formation of a complex between IGFBPs and vitronectin.

[0137] An example of an agonist contemplated by the present invention isIGF-I engineered to include a heparin binding domain (HBD) of an IGFBPto thereby bind vitronectin directly. For example, the sequenceSSSRRAPQT in the C-domain of IGF-I may be engineered to have a BXBBBmotif. A preferred BXBBB motif is the KGRKR sequence of IGFBP-3(residues 228-232).

[0138] Alternatively, the putative vitronectin-binding domain of IGF-II:RVSRRSR (residues 34-40) may be introduced into IGF-I.

[0139] An example of an antagonist of the invention is an IGFBPengineered to mutate basic residues in the HBD (as hereinbeforedescribed) to reduce or prevent binding of the IGFBP to vitronectin.

[0140] Suitably, the engineered IGFBP is capable of binding IGF-I.

[0141] Preferably, the engineered IGFBP is IGFBP-3 or IGFBP-5.

[0142] It is also contemplated that an analogue of an IGFBP could beengineered which enables formation of a complex between the analogue andVN. Suitably, the analogue would also bind an IGF. Potential advantagesof such an analogue is that it might be more readily synthesized orisolated than an IGFBP, have a particular desired biological half-lifeand perhaps be engineered to specifically bind IGF-I.

[0143] The aforementioned mimetics may be peptides, polypeptides orother organic molecules, preferably small organic molecules, with adesired biological activity and half-life.

[0144] Computer-assisted structural database searching is becomingincreasingly utilized as a procedure for identifying mimetics. Databasesearching methods which, in principle, may be suitable for identifyingmimetics, may be found in International Publication WO 94/18232(directed to producing HIV antigen mimetics), U.S. Pat. No. 5,752,019and International Publication WO 97/41526 (directed to identifying EPOmimetics), each of which is incorporated herein by reference.

[0145] Other methods include a variety of biophysical techniques whichindentify molecular interactions. These allow for the screening ofcandidate molecules according to whether said candidate molecule affectsformation of IGF-IGFBP-VN complexes, for example. Methods applicable topotentially useful techniques such as competetive radioligand bindingassays (see Upton et al., 1999, supra for a relevant method), analyticalultracentrifugation, microcalorimetry, surface plasmon resonance andoptical biosensor-based methods are provided in Chapter 20 of CURRENTPROTOCOLS IN PROTEIN SCIENCE Eds. Coligan et al., (John Wiley & Sons,1997) which is incorporated herein by reference.

[0146] Pharmaceutical Compositions

[0147] The invention includes administration of a protein complex of theinvention in the form of a pharmaceutical composition. Pharmaceuticalcompositions of the invention may include isolated protein complexesthat comprise variant IGFs and/or IGFBPs or agents that disrupt orprevent formation of said complexes as hereinbefore described.

[0148] Suitably, the pharmaceutical composition comprises apharmaceutically-acceptable carrier. Pharmaceutical compositions mayalso include polypeptide variants, fragments or mimetics as hereinbeforedefined.

[0149] By “pharmaceutically-acceptable carrier” is meant a solid orliquid filler, diluent or encapsulating substance that may be safelyused in systemic administration. Depending upon the particular route ofadministration, a variety of carriers, well known in the art may beused. These carriers may be selected from a group including sugars,starches, cellulose and its derivatives, malt, gelatine, talc, calciumsulfate, vegetable oils, synthetic oils, polyols, alginic acid,phosphate buffered solutions, emulsifiers, isotonic saline, andpyrogen-free water.

[0150] Any suitable route of administration may be employed forproviding a patient with the composition of the invention. For example,oral, rectal, parenteral, sublingual, buccal, intravenous,intra-articular, intra-muscular, intra-dermal, subcutaneous,inhalational, intraocular, intraperitoneal, intracerebroventricular,transdermal and the like may be employed. Intra-muscular andsubcutaneous injection is appropriate, for example, for administrationof immunogenic compositions, vaccines and DNA vaccines.

[0151] Dosage forms include tablets, dispersions, suspensions,injections, solutions, syrups, troches, capsules, suppositories,aerosols, transdermal patches and the like. These dosage forms may alsoinclude injecting or implanting controlled releasing devices designedspecifically for this purpose or other forms of implants modified to actadditionally in this fashion. Controlled release of the therapeuticagent may be effected by coating the same, for example, with hydrophobicpolymers including acrylic resins, waxes, higher aliphatic alcohols,polylactic and polyglycolic acids and certain cellulose derivatives suchas hydroxypropylmethyl cellulose. In addition, the controlled releasemay be effected by using other polymer matrices, liposomes and/ormicrospheres.

[0152] Pharmaceutical compositions of the present invention suitable fororal or parenteral administration may be presented as discrete unitssuch as capsules, sachets or tablets each containing a pre-determinedamount of one or more therapeutic agents of the invention, as a powderor granules or as a solution or a suspension in an aqueous liquid, anon-aqueous liquid, an oil-in-water emulsion or a water-in-oil liquidemulsion.

[0153] With regard to pharmaceutical compositions comprising IGF andIGFBPs, particular reference is made to U.S. Pat. No. 5,936,064 andInternational Publications WO99/62536 and WO99/54359 which areincorporated herein by reference.

[0154] Pharmaceutical compositions of the invention may also includeexpression vectors such as viral vectors such as vaccinia, and viralvectors useful in gene therapy. The latter include adenovirus andadenovirus-associated viruses (AAV) such as described in Braun-Falco etal.,1999, Gene Ther. 6 432, retroviral and lentiviral vectors such asdescribed in Buchshacher et al., 2000, Blood 95 2499 and vectors derivedfrom herpes simplex virus and cytomegalovirus. A general overview ofviral vectors useful in endocrine gene therapy is provided in Stone etal., 2000, J. Endocrinol. 164 103.

[0155] The present invention may also utilize specific expressionvectors which target gene expression to epidermal cells, such asdescribed in U.S. Pat. No. 5,958,764 and for in vivo wound healingapplications, such as described in U.S. Pat. No. 5,962,427.

[0156] Each of the aforementioned publications is incorporated herein byreference.

[0157] Therapeutic Uses

[0158] The invention provides methods of treatment using polypeptidecomplexes of the invention. These methods are particularly aimed attherapeutic treatment of mammals, and more particularly, humans.

[0159] Such methods include administration of pharmaceuticalcompositions as hereinbefore defined, and may be by way of microneedleinjection into specific tissue sites, such as described in U.S. Pat. No.6,090,790, topical creams, lotions or sealant dressings applied towounds, burns or ulcers, such as described in U.S. Pat. No. 6,054,122 orimplants which release the composition such as described inInternational Publication WO99/47070.

[0160] Gene therapy is also applicable in this regard, such as accordingto methods set forth in U.S. Pat. No. 5,929,040 and U.S. Pat. No.5,962,427.

[0161] There also exist methods by which skin cells can be geneticallymodified for the purpose of creating skin substitutes, such as bygenetically engineering desired growth factor expression (Supp et al.,2000, J. Invest. Dermatol. 114 5). An example of a review of this fieldis provided in Bevan et al., Biotechnol. Gent. Eng. Rev. 16 231.

[0162] Also contemplated is “seeding” a recipient with transfected ortransformed cells, such as described in International PublicationWO99/11789.

[0163] These methods can be used to stimulate cell proliferation andthereby facilitate or progress wound and burn healing, repair of skinlesions such as ulcers, tissue replacement and grafting such as by invitro culturing of autologous skin, re-epithelialization of internalorgans such as kidney and lung and repair of damaged nerve tissue.

[0164] Skin replacement therapy has become well known in the art, andmay employ use of co-cultured epithelial/keratinocyte cell lines, forexample as described in Kehe et al., 1999, Arch. Dermatol. Res. 291 600or in vitro culture of primary (usually autologous) epidermal, dermaland/or keratinocyte cells. These techniques may also utilize engineeredbiomaterials and synthetic polymer “scaffolds”.

[0165] Examples of reviews of the field in general are provided inTerskikh & Vasiliev, 1999, Int. Rev. Cytol. 188 41 and Eaglestein &Falanga, 1998, Cutis 62 1.

[0166] More particularly, the production of replacement oral mucosauseful in craniofacial surgery is described in Izumi et al., 2000, J.Dent. Res. 79 798. Fetal keratinocytes and dermal fibroblasts can beexpanded in vitro to produce skin for grafting to treat skin lesions,such as described in Fauza et al., J. Pediatr. Surg. 33 357, while skinsubstitutes from dermal and epidermal skin elements cultured in vitro onhyaluronic acid-derived biomaterials have been shown to be potentiallyuseful in the treatment of burns (Zacchi et al., 1998, J. Biomed. Mater.Res. 40 187).

[0167] Another aspect of epithelial cell therapy relates to healing ofthe epithelial lining of the gastrointestinal tract to treat or preventimpaired gut function.

[0168] Polymer scaffolds are also contemplated for the purpose offacilitating replacement skin engineering, as for example described inSheridan et al., 2000, J. Control Release 14 91 and Fauza et al., 1998,supra, as are microspheres as agents for the delivery of skin cells towounds and burns (LaFrance & Armstrong, 1999, Tissue Eng. 5 153).

[0169] The aforementioned techniques may be readily utilized accordingto the present invention by use of isolated protein complexes of theinvention to promote skin cell proliferation for the purposes of tissuereplacement and for cosmetic skin treatments.

[0170] With regard to bone regeneration, the invention provides surgicalor prosthetic implants coated, impregnated or otherwise pretreated withan isolated protein complex of the invention.

[0171] Conversely, inhibition or suppression of cell proliferation andmigration by preventing or disrupting formation of IGF-IGFBP-VNcomplexes may constitute a prophylactic or therapeutic treatment ofpsoriasis or malignancies such as epithelial cancers such as breastcancer.

[0172] The invention also contemplates a method of differentiating astem or progenitor cell by administering an isolated protein complex ofthe invention to said stem or progenitor cell. Isolated proteincomplexes comprising variant growth factors and IGFBPs may also beapplicable to this method.

[0173] Differentiated cells produced according to this method may beuseful in therapeutic methods such as as hereinbefore described.

[0174] For example, smooth muscle cells are considered to be“mesenchymal stem cells” and may be driven to differentiate intofibroblasts, stromal cells, endothelial cells, bone or adipocytes.

[0175] In order that the invention may be readily understood and putinto practical effect, particular preferred embodiments will now bedescribed by way of the following non-limiting examples.

[0176] All competitive radioligand binding assays as described hereinwere performed essentially as described in Upton et al., 1999, supra.

EXAMPLE 1

[0177] Competition Binding Assay to Assess Ability of Insulin,Pro-IGF-II and IGF-I to Compete With IGF-II for Binding to Vitronectin

[0178] Due to the similarity in structure shared between IGFs andinsulin, binding of insulin to vitronectin was examined. Crosslinkingexperiments performed by Upton et al., 1999, supra indicated thatinsulin was unlikely to compete with IGF-II for binding to vitronectin,as was the case with IGF-I. As a consequence, high concentrations ofinsulin were examined in order to establish whether insulin couldcompete with radiolabelled IGF-II for binding to vitronectin. Theresults shown in FIG. 1 indicated that, as for the situation originallyobserved with IGF-I (Upton et al., 1999, supra), insulin competed poorlywith [¹²⁵I]-IGF-II for binding to vitronectin.

[0179] Investigation of IGF-I as a competitor for IGF-II binding tovitronectin revealed that IGF-I, could compete with the binding of[¹²⁵I]-IGF-II to vitronectin (FIG. 2). However, IGF-I was markedly lesseffective than IGF-II in competing with radiolabelled IGF-II for bindingto vitronectin, requiring an approximate 3000 fold increase in IGF-Iconcentration above IGF-II to achieve the same effect.

[0180] Similar studies demonstrated that proIGF-II could compete with[¹²⁵I]-IGF-II for binding to vitronectin (FIG. 3). ProIGF-II was not aseffective as IGF-II in competing with [¹²⁵I]-IGF-II for binding tovitronectin with IC₅₀ values of 65.6 nM and 9.6 nM respectively. Thus,the presence of the E domain within proIGF-II represents a structuralmodification that alters the binding to vitronectin. This could be dueto steric hindrance by the additional domain in proIGF-II compared toIGF-II, or may be a result of an alterted structure of the proIGF-IImolecule that alters the affinity of proIGF-II for vitronectin.

EXAMPLE 2

[0181] Investigation of PAI-1 and uPAR as Competitors for Binding ofIGF-II to Vitronectin

[0182] PAI-1 and suPAR were investigated as molecules which mightcompete with IGF-II for binding to vitronectin, as these proteins havebeen reported to bind vitronectin (Declerck et al., 1988, J. Biol. Chem.263 15454; Wei et al.,1994, J. Biol. Chem. 269 32380; Kanse et al.,1996, Exp. Cell Res. 224 344). At concentrations up to 2000 nMcompetition of PAI-1 with ¹²⁵I-IGF-II for binding to vitronectin wasobserved with an approximate IC₅₀ value of 524 nM (FIG. 4). While thisconcentration is relatively high in terms of IC₅₀ values, suchconcentrations can be found in vivo associated with tumours(Grondahl-Hansen et al.,1993, Cancer Res. 53 2513). Indeed, Kjoller etal., 1997, Exp. Cell Res. 232 420, found 370 nM of PAI-1 was required toachieve half maximal effect in an assay assessing the ability of PAI-1to inhibit cell migration of WISH cells. This inhibition was postulatedto result via PAI-1 competing with integrins and uPAR for binding tovitronectin. Thus, the observed interaction between PAI-I, IGF-II andvitronectin may indeed have some functional consequences in vivo,particularly when IGF-II and PAI-1 levels are highly expressed as issometimes found in tumours.

[0183] While it is known that PAI-1 binds to the N-terminal somatomedinB domain of vitronectin, the high concentrations of PAI-1 required toeffectively compete with IGF-II for binding to vitronectin suggestedthat these studies have not provided any clear information regarding thelocation of IGF-II of the binding site on vitronectin. However, theresults may be interpreted two ways. The first possibility is that theobserved competition between IGF-II and PAI-1 is due to partialcompetition at the primary high affinity site on vitronectin within thesomatomedin B domain via steric hindrance. Alternatively, thecompetition could be due to direct competition between IGF-II and PAI-1,for binding to a site on vitronectin to which PAI-1 binds with a reducedaffinity. Further experimental studies are required to clarify thisissue.

[0184] While the present inventors were unable to show that solublesuPAR competes for binding of IGF-II to VN, this may have been due tothat fact that lyophilized suPAR was used after internationaltransportation. There is no evidence that reconstituted suPAR isbiologically active after reconstitution, although the observation that[¹²⁵I-suPAR did not bind VN (data not shown) perhaps supports aninterpretation that the suPAR used in these studies was inactive.

[0185] Use of similar techniques to those that identified the PAI-1binding site on vitronectin to be within a fragment of vitronectincontaining the 44 N-terminal amino acids (Deng et al., 1995, Thromb.Haemost. 74 66), may establish whether the competition observed betweenPAI-1 and IGF-II for binding vitronectin is a result of either of thesepossibilities.

[0186] It has previously been shown that the soluble form of theurokinase receptor (suPAR) can bind to immobilized vitronectin (Wei etal., 1994, supra). In the studies reported here, however, even atconcentrations up to 300 nM, no competition between suPAR and[¹²⁵I]-IGF-II for binding to vitronectin was observed.

EXAMPLE 3

[0187] Binding of IGF-I and IGF-II to Vitronectin in the Presence ofNon-Glycosylated Recombinant IGFBP-3

[0188] To investigate whether IGFBPs could mediate binding of the IGFsto vitronectin, the effect of increasing concentrations of IGFBP-3 inthe presence of [¹²⁵I]-IGF-I or [¹²⁵I]-IGF-II was assessed. The resultsare shown in FIG. 5.

[0189] At the concentrations tested, 10 ng of IGFBP-3 per well causedthe greatest amount of [¹²⁵I]-IGF-I to bind to vitronectin coated wells,whereas 30 ng of IGFBP-3 per well caused the greatest amount of[¹²⁵I]-IGF-II to bind to vitronectin coated wells. The effect was mostnoticeable in the case of [125I]IGF-I, since low counts of [125I]-IGF-Ibound to vitronectin, and even at the lowest concentration of IGFBP-3(3.7 ng), this binding was increased (FIG. 5A). Conversely,[¹²⁵I]-IGF-II did not show an increase over binding obtained onvitronectin coated wells alone, until concentrations of 11 ng/100 μL-33ng/100 μL were reached (FIG. 5B).

[0190] Preincubation of IGFBP-3 with either [¹²⁵I]-IGF-I or[¹²⁵I]-IGF-II did not alter the binding to vitronectin as compared tothe non pre-incubated concentrations of IGFBP-3 and [¹²⁵I]-IGF-I or[¹²⁵I]-IGF-II.

EXAMPLE 4

[0191] Binding of IGF-I to Vitronectin in the Presence of RecombinantIGFBPs Produced in Mammalian Cells

[0192] Binding assays examining the ability of [¹²⁵I]IGF-I to bind toVN-coated dishes in the presence of IGFBPs, added at the same time asthe radiolabel, were performed as described in Upton et al.,1999, supra.The data are shown in FIG. 6. Increasing amounts of IGFBP-2, -4 and -5resulted in increased binding of labelled IGF-I to vitronectin coatedwells. At the highest amount of IGFBP tested, 5 ng, binding of labelledIGF-I was increased approximately 2.8-, 3.8- and 8-fold for IGFBP-2, 4and 5 compared to control wells where VN, but no IGFBPs, were present.

[0193] The presence of IGFBP-3 at 0.05, 0.2 and 0.5 ng/well alsoincreased binding of labelled IGF-I to VN while 2 and 5 ng ofIGFBP-3/well appeared to inhibit binding of the radiolabel. Allconcentrations of IGFBP-1 and -6 tested were also inhibitory.

[0194] These results demonstrate that unlike the situation with IGF-II,minimal direct binding of IGF-I to VN is observed. However, the presenceof IGFBPs, especially IGFBP-2, -3, -4 and -5 enhances IGF binding toVN-coated wells, suggesting that IGFBPs mediate the binding of IGF-I toVN in this situation. In addition, the data suggests that IGFBPs havethe potential to both enhance and inhibit binding of IGF-I to VN,depending on a) which IGFBPs are present and b) the amount of IGFBPpresent.

EXAMPLE 5

[0195] Binding of Labelled IGF-I to VN in the Presence IGFBP-3 Variants

[0196] Binding assays examining the ability of [¹²⁵I]IGF-I to bind toVN-coated dishes in the presence of an IGFBP-3 variant in which theputative “heparin binding domain” was mutated (IGFBP-3 HBD) and avariant in which glycosylation sites had been mutated (Non-gly IGFBP-3)were performed as described above. The data are shown in FIG. 7.

[0197] The IGFBP-3 glycosylation mutant used in these binding studieshad three potential O-glycosylation sites at positions Asn89, Asn109,Asn172 mutated to Ala.

[0198] The IGFBP-3 HBD mutant did not enhance binding of [¹²⁵I-IGF-I toVN-coated dishes, suggesting the heparin binding domain of IGFBP-3 isinvolved in binding IGFBP-3 to VN. Other studies have identified thatamino acid residues outside of the putative “heparin binding domain” areresponsible for IGF-I binding to IGFBP-3, hence it is most likely thatthe results represent decreased binding of IGFBP-3 to VN, rather thandecreased binding of labelled IGF-I to IGFBP-3 (Imai et al., 2000, JBiol Chem 275:18188-18194).

[0199] Interestingly, the non-glycosylated IGFBP-3 mutant significantlyenhanced binding of [¹²⁵I]-IGF-I to bind to VN-coated dishes. Thestriking 20-fold increase in binding of labelled IGF-I to VN-coatedwells in the presence of 2 ng of the mutant IGFBP-3 is intriguing andsuggests that glycosylation of IGFBP-3 inhibits interaction of either a)IGF-I to IGFBP-3 or b) IGFBP-3 with VN. Alternatively, both interactionsmay be hindered by the presence of carbohydrates.

[0200] Whether non-glycoslyated IGFBP-3 is functionally relevant in vivoremains to be established. Nevertheless, this finding suggests thatnon-glycosylated IGFBP-3 bound to VN may be a useful way to deliverIGF-I to sites where IGF-I is required to potentiate cell function suchas in stimulating cell proliferation. Alternatively, non-glycosylatedIGFBP-3 bound to VN may provide a mechanism for sequestering excessIGF-I in situations where cell proliferation is not required such as intumours overexpressing IGFs.

EXAMPLE 6

[0201] Competition Binding Assay Assessing Ability of IGF Variants toCompete With Labelled IGFs for Binding to VN in the Presence ofNon-Glycosylated Recombinant IGFBP-3

[0202] The concentration of IGFBP3 that produced the highest binding ofradiolabelled IGF depicted in FIG. 5 was used in a competition assay todetermine whether increasing concentrations of IGFs and desIGFs couldcompete with radiolabelled IGFs for binding to vitronectin in thepresence of IGFBP3. The results indicated that IGF-I, IGF-II,des(1-6)IGF-II and perhaps also des(1-3)IGF-I (not shown), at highconcentrations, can compete with the binding of [¹²⁵I]-IGF-II tovitronectin in the presence of 10 ng of IGFBP3 (FIG. 8). Theseexperimental results indicated that only IGF-I, -II and des(1-6) IGF-IIcould compete with IGF-II for binding to vitronectin coated wells in thepresence of 30 ng of IGFBP3.

EXAMPLE 7

[0203] Stimulation of Cell Proliferation by Isolated Protein ComplexesComprising IGF-II and Vitronectin

[0204] The strategy of pre-binding IGF-II to VN was used in this studyin an attempt to more accurately reflect the extracellular environmentin vivo. Most cell culture approaches add exogenous substrates insolution phase; thereby the cells are constantly exposed to thetreatments. Cells in tissues do not encounter this “constant, solutionphase” environment in vivo. Thus the approach adopted for this study wasto pre-bind IGFs to VN, a situation which more accurately mirrors the invivo conditions.

[0205] Following pre-binding of IGFs to VN in culture dishes, cells wereseeded into wells and the ability of IGFs complexed to VN to stimulateprotein synthesis was examined by established methods (Francis et al.,1986, Biochem J. 233:207-213). Increased protein synthesis correlateswith increases in cell number, hence is a reflection of cellproliferation. Responses, expressed as percentage above control wells inwhich no VN or IGFs were present, measured the incorporation of[³H]-leucine into newly synthesised protein over 48 hrs.

[0206] Referring to the data in FIG. 9, when 3, 10, 30, 100, 300 and1000 ng of IGF-II was pre-bound to the wells in the absence of VN,resulting responses of 8, 12, 10, 16, 24 and 43% above the control wells(-VN, -IGF) respectively were observed. Furthermore, these same doses ofIGF-II pre-bound to VN coated wells stimulated incorporation of[³H]-leucine into protein with effects of 19, 29, 39, 51, 70 and 101%respectively. Combining the responses obtained with VN alone (12%) withthat obtained for IGF-II alone (refer to above) gives rise to predictedadditive effects of 20, 24, 22, 28, 36 and 55% for 3, 10, 30, 100, 300and 100 ng of IGF-II respectively. These values are significantlydifferent (p<0.05) to the actual effects observed when IGF-II waspre-bound to VN at all doses except for the two lowest amounts of IGF-IItested (3 and 10 ng). Thus IGF-II pre-bound to VN stimulates synergisticeffects in protein synthesis ranging from 5 to 46% greater than thecalculated additive effects. These responses may well be a result of thedirect binding of IGF-II to VN, and may also arise from indirect bindingof IGF-II to VN via IGFBPs. HaCAT keratinocytes produce large amounts ofIGFBP-3 (Wraight et al., 1994, J. Invest. Dermatol 103:627-631).

EXAMPLE 8

[0207] Binding of Labelled IGF-II to VN in the Presence of RecombinantIGFBPs Produced in Mammalian Cells

[0208] Binding assays examining the ability of [¹²⁵I]IGF-II to bind toVN-coated dishes in the presence of IGFBPs, added at the same time asthe radiolabel, were performed as described in Upton et al., 1999,supra. The IGFBPs used in these studies were glycosylated having beenproduced in mammalian cells. As shown in FIG. 10, increasing amounts ofIGFBP-1, -3 and -6 resulted in decreased binding of labelled IGF-II tovitronectin coated wells in a dose-dependent manner. IGFBP-2 alsoappeared to compete for binding of labelled IGF-I to VN, albeit lesseffectively than IGFBP-1, -3 or -6. IGFBP-4 on the other hand had littleeffect on binding of IGF-II to VN, while IGFBP-5 appeared to enhanceIGF-II binding to a small extent. The inhibitory effect of IGFBP-3 couldresult from IGFBP-3 competing for binding of IGF-II to the same bindingregion on VN. Alternatively, or in addition, the inhibitory effect ofIGFBP-3, as well as IGFBP-1 and -6. may arise from the affinity ofIGF-II for VN being less than the affinity of IGF-II for these IGFBPs,hence these IGFBPs sequester IGF-II and the complex does not bind to VN.

EXAMPLE 9

[0209] Stimulation of Cell Proliferation by Isolated Protein ComplexesComprising IGF-I, IGFBP-5 and Vitronectin

[0210] Referring to Table 2, IGF-I with IGFBP-5 and vitronectinstimulated keratinocyte proliferation (as measured by ³H-leucineincorporation into newly synthesized protein) at all concentrationstested with synergistic effects observed at the highest amount tested.

[0211] It is proposed by the present inventors that the effect ofisolated protein complexes upon cell proliferation may be even greaterat higher concentrations of IGFBP-5 than the relatively low amountdescribed in Table 2.

EXAMPLE 10

[0212] Engineering IGF and IGFBP Variants

[0213] Amino acid residues in IGFBP-3 that are important for associationwith the ECM and have been defined as the putative “heparin-bindingdomain” are residues KGRKR at positions 228-232. Similar residues arefound in the corresponding region of IGFBP-5. The IGFBP-3 HBD mutantthat was used in the binding studies reported herein had the residuesKGRKR altered to MDGEA based on the amino acids found in thecorresponding positions in IGFBP-1. These changes result in a chargereversal in this part of the protein. This mutant still binds IGF-I andIGF-II with high affinity but binds the acid-labile subunit and the cellsurface poorly (Firth et al., 1998, J. Biol. Chem. 273 2631-2638).

[0214] Heparin binding motifs in a diverse range of proteins wereoriginally described in Cardin et al., 1989, Arteriosclerosis 9 21-32.

[0215] The C-domain of human IGF-II contains a number of positivelycharged amino acid residues and in particular positions 34-40 containsthe amino acids RVSRRSR. Given that positively charged amino acids areimportant in mediating binding IGFBPs to cell surfaces and to VN, theseamino acids in IGF-II may be important in binding of IGF-II directly toVN. Regardless, it would be a relatively simple procedure to introduce a“heparin binding motif” similar to that found in IGFBP-3 (BXBBB; where Bis a basic amino acid) by creating an IGF-II mutant with deletions ofeither V³⁵ or S³⁶ along with S³⁹. The importance of positive residues inmediating binding of IGF-II to VN is further illustrated by the presentinventors' evidence of reduced binding of the chicken IGF-II mutant,(desR⁴⁰)-IGF-II, to VN.

[0216] The C-domain of IGF-I on the other hand contains a relativelynon-charged stretch of amino acids in the corresponding region of theprotein to that described above for IGF-II. This may explain why IGF-Idoes not bind directly to VN. In addition, insulin, which also does notbind to VN (or to IGFBPs) does not have a corresponding C-domain as itis cleaved out in the mature protein. Human IGF-I SSSRRAPQT Human IGF-IIRVSRRS- - R

[0217] Introduction of the IGF-II sequence RVSRRSR or the IGFBP3sequence KGRKR into IGF-I could enable IGF-I to bind VN directly.

EXAMPLE 11

[0218] Isolated Protein Complexes, Cell Proliferation and Survival

[0219] Bcl-2 transcription, a critical element of the cell survivalpathway, is elevated in cells that attach to VN through alphav-beta3integrins. (Matter & Ruoslahti, 2001, J. Biol. Chem. 276 27757-27763).In addition, IGF-I protects cells from apoptosis by elevating bcl-2transcription in an AKT-dependent manner (Pugazhenthi et al., 1999, JBiol. Chem. 274 27529-35). The IGF receptor physically associates withthe alphav-beta3 integrin with a synergistic effect on cell growth(Schneller et al., 1997, EMBO J 16 5600-5607). Thus isolated proteincomplexes of the invention may provide an extracellular point ofintegration for initiating the cell survival signals mediated by boththe integrin and the growth factor receptor.

[0220] IGFBP-5 has been demonstrated to potentiate the anti-apoptoticand mitogenic effects of IGF-I in prostate cancer cells (Miyake et al.,2000, Endocrinol. 141 2257-2265). In addition, the synthesis of VN invivo by glioma cells and in colorectal adenocarcinoma correlates withtumour grade (Uhm et al., 1999, Clin Cancer Res. 5 1587-1594;Tomasini-Johansson et al., 1994, Exp Cell Res. 214 303-312; Gladson etal., 1995, J. Cell Sci. 108 947-56; Gladson & Cheresh, 1991, J. Clin.Invest. 88 1924-32).

[0221] Hence, according to the present invention, it is proposed thatIGF:IGFBP:VN complexes formed in vivo may promote tumour cell survivaland progression. The invention therefore contemplates therapeutic agentsthat disrupt in vivo complex formation.

[0222] The heparin-binding domain of VN has been reported to inhibitfibronectin matrix assembly (Hocking et al., 1999, J. Biol. Chem. 27427257-27264). Reduced fibronectin deposition is associated with tumourcell invasion as decreased cell migration rates are associated withincreased levels of polymerised fibronectin (Morla et al., 1994, Nature367 193-196). Hence, IGP:IGFBP complexes bound to the heparin bindingdomain of VN, may dampen fibronectin matrix assembly and facilitatetumour invasion of local connective tissue. Thus, the inventioncontemplates therapeutic agents that disrupt these ill vivo complexes toreduce tumour invasiveness.

[0223] Isolated Protein Complexes and Wound Healing

[0224] The converse argument can be used to support the use of thecomplex in situations where cell migration is required such as in woundrepair. The IGF system plays an important role in wound healing and bothIGF-I and IGFBP-3 are present in wound fluid in significantconcentrations. (Skottner et al., 1990, Acta Scand. Suppl. 367 63-66;Clark R (ed) 1996, Molecular and Cell Biology of Wound Repair, pp 3-50,Plenum Press, New York; Robertson et al., 1996, Endocrinol. 1372774-2784; Vogt et al.,1998, Growth Horm. IGF Res. 8 Suppl B:107-9.

[0225] IGFBPs have been demonstrated to reduce the rate of IGF clearancefrom wounds. (Robertson et al., 1999, Am J Physiol. 276 E663-71).IGFBP-3:IGF-I complexes bind to fibrin clots ill vitro leading to thesuggestion this also occurs in vivo, resulting in concentration of IGF-Iat wound sites. (Campbell et al., 1999, J. Biol. Chem. 274 30215-30221).Similarly, vitronectin binds to fibrin (Podor et al., 2000, J. Biol.Chem. 275 19788-19794). It is also noted that vironectin-null miceexhibit increased wound fibrinolysis and decreased microvascularangiogenesis (Jang et al, 2000, Surgery 127 696-704).

[0226] According to the present invention, it is proposed that IGFsbound to IGPBPs can bind to VN, which in turn associates with the fibrinclot, thus providing a reservoir of IGFs at the wound site. Thusisolated protein complexes of the invention could be administered towounds to accelerate the repair process.

[0227] A particular aspect of would healing contemplated by the presentinvention relates to healing diabetic foot ulcers. Wound healing isdelayed in diabetes. Growth factors influence the healing process and inparticular, IGFs have been shown to stimulate keratinocyteproliferation. However, analysis of tissues from diabetic skin and footulcers reveals lack of expression of IGF-I within the basal layer andfibroblasts compared to tissue sections from non-diabetic patients.(Blakytny et al., 2000, J. Pathol. 190 589-594). Isolated proteincomplexes of the invention could be useful in delivery of IGF-I to thesetypes of wounds.

[0228] Isolated Protein Complexes and Bone Engineering

[0229] IGFBP-5 facilitates binding of labelled IGF-I to bone by amechanism that is independent of IGF receptors. (Mohan et al., 1995, J.Biol. Chem 270 20424-20431) and enhances IGF-stimulated osteoblastfunction. (Andress, 1995, J. Biol. Chem. 270 28289-28296).

[0230] The implant material hydroxylapatite has been shown in numerousstudies to be highly biocomaptible and to osseointegrate well withexisting bone. Recent evidence has found that hydroxylapatite willabsorb more VN from serum than other commonly used implant materialssuch as titanium and stainless steel. The absorption of VN wasaccompanied by greater binding of osteoblast precursor cells (Kilpadi etal., 2001, J. Biomed. Mater. Res. 57 258-267).

[0231] The effect of VN on nanophase alumina vs conventional alumina wasrecently examined with VN being found to enhance osteoblast adhesion(Webster et al., 2001, Tissue Eng 7 291-301).

[0232] The present inventors propose that isolated protein complexes ofthe invention may useful as coating applied to these materials andthereby accelerate bone cell attachment, growth and integration inorthopaedic applications such as hip joint replacements.

[0233] VN enhances IGF-I stimulated osteoclastic resorption andproteinase activities in rabbit bone cell culture. (Rousselle et al.,2001, Histology & Histopathology 16 727-734) and IGFBP-5 enhancesIGF-stimulated osteoblast mitogenesis. (Andress & Birnbaum, 1992, J.Biol. Chem. 267 22467-22472).

[0234] The present inventors propose that as the major IGFBP produced bybone cells is IGFBP-5, the potentiation of the IGF effect by VN islikely to also involve IGFBP-5.

[0235] Isolated Protein Complexes and Treatment of Atherosclerosis

[0236] IGF-I has previously been implicated in the development ofexperimental atherosclerotic lesions. Moreover, alphav-beta3 inhibitorshave been demonstrated to reduce atherosclerotic lesions—this beingassociated with inhibition of IGF-I-mediated signaling. (Nichols et al.,1999, Circ. Res. 851040-1045).

[0237] Given that:

[0238] (i) IGFBP-5 and VN are synthesised and secreted by arterialsmooth muscle cells and are present in blood vessel walls; and

[0239] (ii) IGF:IGFBP-5:VN complexes promote vascular smooth muscle cellmitogenesis and migration (Nam & Clemmons, 2000, Growth Horm. IGF Res.10:A23);

[0240] it is proposed by the present inventors that IGF:IGFBP-5:VNcomplexes are involved in formation of atherosclerotic lesions. Thustherapeutic agents that disrupt complex formation may hold potential intreating atheroslerosis.

[0241] Throughout the specification the aim has been to describe thepreferred embodiments of the invention without limiting the invention toany one embodiment or specific collection of features. It will thereforebe appreciated by those of skill in the art that, in light of theinstant disclosure, various modifications and changes can be made in theparticular embodiments exemplified without departing from the scope ofthe present invention.

[0242] It will also be appreciated that all patent and scientificliterature and computer programs referred to are incorporated herein byreference. TABLE 1 Nucleic acid Reference IGF-I Jansen et al., 1983,Nature 306 609 IGF-II Jansen et al., 1985, FEBS Lett 179 243 IGFBP-1Brinkman et al., 1988, EMBO J. 7 2417 IGFBP-2 Binkert et al., 1989, EMBOJ. 8 2497 IGFBP-3 Wood et al., 1988, Mol. Endocrinol. 2 1176 IGFBP-4LaTour et al., 1990, Mol. Endocrinol. 4 1806 IGFBP-5 Kiefer et al.,1991, Biochem. Biophys. Res. Comm. 176 219 IGFBP-6 Shimasaki et al.,1991, Mol. Endocrinol. 5 938 ALS Leong et al., 1992, Mol. Endocrinol. 6870 Vitronectin Suzuki et al., 1985, EMBO J. 4 2519

[0243] TABLE 2 Treatment −Vitronectin +Vitronectin Control 100 ± 2.6 —IGFBP-5 — 109.5 ± 9.2 100 ng IGF-1 + IGFBP-5 117.4 ± 10.2 119.4 ± 1.9300 ng IGF-1 + IGFBP-5 140.9 ± 2.0 154.3 ± 1.8 1000 ng IGF-1 + IGFBP-5144.3 ± 11.0 161.4 ± 9.1

1. An isolated protein complex comprising a growth factor bindingprotein and vitronectin.
 2. The isolated protein complex of claim 1,further comprising a growth factor.
 3. The isolated protein complex ofclaim 2, wherein the growth factor is insulin like growth factor-I(IGF-I).
 4. The isolated protein of claim 2, wherein the growth factoris selected from the group consisting of epidermal growth factor (EGF),fibroblast growth factor (FGF), basic fibroblast growth factor (bFGF),osteopontin, thrombospondin-1, tenascin-C, PAI-1, plasminogen,fibrinogen, fibrin and transferrin.
 5. The isolated protein complex ofclaim 1, wherein the growth factor binding protein is selected from thegroup consisting of IGFBP1, IGFBP2, IGFBP3, IGFBP4, IGFBP5 and IGFBP6.6. The isolated protein complex of claim 5, wherein the insulin-likegrowth factor binding protein is IGFBP2, IGFBP3, IGFBP4 or IGFBP5. 7.The isolated protein complex of claim 6, wherein the insulin-like growthfactor binding protein is IGFBP3 or IGFBP5.
 8. An isolated proteincomplex comprising an IGFBP-related protein and vitronectin.
 9. Theisolated protein complex of claim 8, further comprising a growth factor.10. This isolated protein complex of claim 9, wherein the growth factoris IGF-I or IGF-II.
 11. The isolated protein complex of claim 10,wherein the IGFBP-related protein is selected from the group consistingof: (i) connective tissue growth factor (CTGF); (ii) a polypeptideencoded by the mac25 gene; (iii) a polypeptide encoded by the nov gene;and (iv) a polypeptide encoded by the cyr61 gene.
 12. An isolatedprotein complex comprising vitronectin, a variant growth factor and/or avariant growth factor binding protein.
 13. The isolated protein complexof claim 12, comprising vitronectin and a variant growth factorengineered to include a heparin binding domain (HBD).
 14. The isolatedprotein complex of claim 13, wherein the variant growth factor is IGF-Iengineered to include said HBD.
 15. The isolated protein complex ofclaim 15, consisting of IGF-I engineered to include said HBD bounddirectly to vitronectin.
 16. The isolated protein complex of claim 13,wherein the HBD has the amino acid sequence BXBBB, wherein B is a basicamino acid residue.
 17. The isolated protein complex of claim 14,wherein the HBD has the amino acid sequence KGRKR.
 18. A variant IGFBPhaving a deleted or mutated IBD, wherein the IGFBP is unable to form theisolated protein complex of claim
 1. 19. The variant IGFBP of claim 19which is capable of binding IGF-P.
 20. Use of the variant of claim 19 asan antagonist.
 21. The isolated protein complex of claim 12, comprisingvitronectin and a non-glycosylated growth factor binding protein. 22.The isolated protein complex of claim 21, wherein the non-glycosylatedgrowth factor binding protein is IGFBP3.
 23. The isolated proteincomplex of claim 13, comprising vitronectin and a variant growth factorselected from the group consisting of des(1-6)IGF-II and des(1-3)IGF-I.24. The isolated protein complex of claim 1, claim 8 or claim 12,wherein vitronectin is multimeric.
 25. The isolated protein complex ofclaim 1, claim 8 or claim 12, wherein vitronectin is monomeric.
 26. Theisolated protein complex of claim 24, wherein each vitronectin monomeris bound to the same or different IGFBP selected from the groupconsisting of: IGFBP1, IGFBP2, IGFBP3, IGFBP4, IGFBP5 and IGFBP6. 27.The isolated protein complex of claim 26, further comprising IGF-IIdirectly bound to vitronectin.
 28. A pharmaceutical compositioncomprising the isolated protein complex of any one of claims 1, 8 or 12and a pharmaceutically-acceptable carrier or diluent.
 29. A surgicalimplant or prosthesis comprising the isolated protein complex of any oneof claims 1, 8, or
 12. 30. A transformed cell capable of expressing arecombinant protein complex according to any one of claims 1, 8 or 12,or recombinant polypeptides capable of forming said complex.
 31. Amethod of modulating cell proliferation and/or migration including thestep of administering to an animal or isolated cells thereof, anisolated protein complex according to any one of claims 1, 8 or
 12. 32.A method of modulating cell proliferation and/or migration, includingthe step of administering to an animal or isolated cells thereof anagent which prevents or disrupts formation of the protein complex of anyone of claims 1, 8 or
 12. 33. The method of claim 32 wherein the agentprevents or disrupts an interaction between an IGFBP and vitronectin.34. The method of claim 32, wherein the agent is IGF-II.
 35. Use ofIGF-II to inhibit formation of the isolated protein complex of claim 1.36. Use of the isolated protein complex of any one of claims 1, 8 or 12for the preparation of a medicament for the treatment of an epithelialcell disease.
 37. Use according to claim 36 wherein the epithelial celldisease is psoriasis or epithelial breast cancer.
 38. Use according toclaim 36, wherein the epithelial cell disease causes impaired gutfunction through deterioration of the gastrointestinal epitheliallining.
 39. Use of the isolated protein complex of any one of claims 1,8 or 12 for the preparation of a medicament for promoting wound healing,skin repair, ulcer and burn healing or in vitro skin regeneration. 40.Use of the isolated protein complex of any one of claims 1, 8 or 12 forthe preparation of a medicament for promoting bone regeneration.
 41. Useof the isolated protein complex of any one of claims 1, 8 or 12 for thepreparation of a medicament for promoting repair of damaged neuronaltissue.
 42. Use of an agent that disrupts or prevents formation of theisolated protein complex of any one of claims 1, 8 or 12 for thepreparation of a medicament for treating cancer.
 43. Use of an agentthat disrupts or prevents formation of the isolated protein complex ofany one of claims 1, 8 or 12 for the preparation of a medicament fortreating atherosclerosis.